synergistic effect in viral-bacterial infection. : 2. influence of viral infection on the phagocytic...

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Acta path. microbiol. scand. Section B. 78, 41 -50, 1970 SYNERGISTIC EFFECT IN VIRAL- BACTERIAL INFECTION 2. Influence of Viral Infection on the Phagocytic Ability of Alveolar Macrophages MIKLOS DEGR~ The Bacteriological Laboratory, Ullevil Hospital, Oslo, Norway The influence of prior virus infection on the antibacterial activity of mouse alveolar niacrophages against Haemophilus influenrue was investigated. Parainfluenza 1 virus in- hibited the phagocytic activity both after in vivo and after in vitro infection. The capacity of macrophages to kill phagocytosed bacteria was not affected by previous virus infection. Similar results were obtained when phagocytosis experiments were conducted with macro- phages in suspension or when cells were attached to solid surfaces. The virus infection had no effect on the opsonic activity of serum. The reduction of phagocytic capacity was most pronounced 24 and 48 hours after virus infection, thereafter it gradually increased to normal level. These data did not correlate with the mortality rate, the reduction of eli- mination of bacteria, and frequency of gross pathological changes of the lung. The extent of these changes was highest when the infectious agents were given 4 days apart. It is questionable therefore whether the antiphagocytic action of the virus upon the alveolar macrophages is the critical factor in the pathogenesis of dual infection. From the present data it is not possible to determine the relative importance of inhibition of phagocytosis in the total reduction of host defence after virus infection. ‘The observation of a synergistic effect of combined respiratory tract infection in the mouse with parainfluenza 1 virus (Sendai strain) and Harniophilus influenzae b, was the subject of a previous report (3). The in- fection was characterized by a significantly enhanced mortality due to pneumonia, as compared with that after infection with either agent alone. Elimination of bacteria was slower in animals previously infected with virus. Presence of high bacterial con- centration in the lung corresponded regularly with clinical illness and with gross patho- logical changes of the lung. No similar as- sociation was demonstrated for the infecting Received 23.vi.69 virus. On the basis of these findings we as- sumed that a critical factor in the devel- opment of dual infection is the local de- fense mechanism by which the intruding foreign particles arc eliminated from the lung. The local host defense mechanism in the respiratory tract is a complex of many fac- tors. Foreign particles may be removed through a haematogeneous route, by the lymph flow, or through the bronchial tree. Viable bacteria may be killed by phagocytic cells, by the bactericidal action of mucus, or by the antibacterial activity of humoral sys- tems. Probably the two most important single factors of these are the mucociliary flow trough the trachea, bronchi and bronctiioli 41

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Page 1: SYNERGISTIC EFFECT IN VIRAL-BACTERIAL INFECTION. : 2. Influence of Viral Infection on the Phagocytic Ability of Alveolar Macrophages

Acta path. microbiol. scand. Section B. 78, 41 -50, 1970

SYNERGISTIC EFFECT IN VIRAL- BACTERIAL INFECTION

2. Influence of Viral Infection on the Phagocytic Ability of Alveolar Macrophages

MIKLOS D E G R ~

The Bacteriological Laboratory, Ullevil Hospital, Oslo, Norway

The influence of prior virus infection on the antibacterial activity of mouse alveolar niacrophages against Haemophilus influenrue was investigated. Parainfluenza 1 virus in- hibited the phagocytic activity both after in vivo and after in vitro infection. The capacity of macrophages to kill phagocytosed bacteria was not affected by previous virus infection. Similar results were obtained when phagocytosis experiments were conducted with macro- phages in suspension or when cells were attached to solid surfaces. The virus infection had no effect on the opsonic activity of serum. The reduction of phagocytic capacity was most pronounced 24 and 48 hours after virus infection, thereafter it gradually increased to normal level. These data did not correlate with the mortality rate, the reduction of eli- mination of bacteria, and frequency of gross pathological changes of the lung. The extent of these changes was highest when the infectious agents were given 4 days apart. I t is questionable therefore whether the antiphagocytic action of the virus upon the alveolar macrophages is the critical factor in the pathogenesis of dual infection. From the present data it is not possible to determine the relative importance of inhibition of phagocytosis in the total reduction of host defence after virus infection.

‘The observation of a synergistic effect of combined respiratory tract infection in the mouse with parainfluenza 1 virus (Sendai strain) and Harniophilus influenzae b, was the subject of a previous report ( 3 ) . The in- fection was characterized by a significantly enhanced mortality due to pneumonia, as compared with that after infection with either agent alone. Elimination of bacteria was slower in animals previously infected with virus. Presence of high bacterial con- centration in the lung corresponded regularly with clinical illness and with gross patho- logical changes of the lung. No similar as- sociation was demonstrated for the infecting

Received 23.vi.69

virus. On the basis of these findings we as- sumed that a critical factor in the devel- opment of dual infection is the local de- fense mechanism by which the intruding foreign particles arc eliminated from the lung.

The local host defense mechanism in the respiratory tract is a complex of many fac- tors. Foreign particles may be removed through a haematogeneous route, by the lymph flow, or through the bronchial tree. Viable bacteria may be killed by phagocytic cells, by the bactericidal action of mucus, or by the antibacterial activity of humoral sys- tems. Probably the two most important single factors of these are the mucociliary flow trough the trachea, bronchi and bronctiioli

41

Page 2: SYNERGISTIC EFFECT IN VIRAL-BACTERIAL INFECTION. : 2. Influence of Viral Infection on the Phagocytic Ability of Alveolar Macrophages

carrying p i ticks towards the pharynx, and the action of alveolar macrophages (21 ).

The importance of phagorytic and bac- tericidal action of alveolar macrophages has been pointed out by 5everal authors (7, 8, 11. 21) . I t is known that a great number of factors, both endogenous and exogenous, m,iy influence phagocyto\is. 'The pulmonary ma- crophage system is highly susceptible to sev- eral factors, P . g. variations in pH, tiypoxia, cortisol, tobacco smoke or cthyl alcohol re- duces its antibacterial capacity ( 11 ) . It has also been reported that different viruse4 may inhibit the phagocytic action of leucocytes (4, 5, 6, 13). The interaction between vi- ruses and macrophages in infected animals is by no means a uniform procedure (14), and the reduction of phagocytosis is not a regularly demonstrated phcnomrnon. It has not been reported previously whether para- influenza virus inhibits this action when in- fecting macrophages.

The aim of the present study was to in- vrstigate the interaction betwern mouse macrophages and parainfluenza virus, with special reference to the effect of infection on the antibacterial properties of the cells.

M A T E R I A 1. S A S D M E T H 0 D S

Mice: HaM/ICR/CSE/Bom white albino strain originally obtained from the National Institute of Public Health, Oslo, was employed. Macrophages were obtained from male mice, 3 5 months old, weighing 22-28 g. For mortality series young adults of either sex were employed. Mice were regularly tested for the presence of haemaggluti- nating virus in their lung and all were negative.

Virus: Parainfluenza 1 virus, Sendai strain, nri- ginally obtained from Dr. E. F . Whee lork , was propagated in the allantoic cavity of embryonated eggs, 9-1 1 days old. The virus pool was stored at -70" C . Virus was titrated i n ernbryonated eggs and by the haemagglutination method, using guinea pig erythrocytes.

Bacteria: Hnemophilus influenzue b 5 1, origi- nally from H. C. Engbzk, Copenhagen, was ob- tained from Dr. T. Omland. T h e strain was used after 42 passages on solid medium. A large number of samples, 1 ml each, were frozen in Lcvinthal broth and kept at -18" C . For phagocytosis ex- periments a sample was incubated in 5 ml of Le- vinthal broth for 18 hours a t 37" C , then cen-

trifuged at 3500 rpm for 10 minutes and recnn- stituted in physiological saline.

Infcrtion of micr: All inoculations were done by the intranasal route under light ether anaes- thesia. 'Thc animals wrre held in an upright po- sition and 0. l nil of fluid containing virus, bac- teria or control fluid was placed in the nasal openings by nieans of a graded 0.1 nil pipette. The animals were held until the fluid disappearrd i n the nasal openings.

Hnrzwsting mncrophoges: Hanks balariced salt solution (BSS) containing 1 per cent Ethylene diamino tetra-acetic acid, dipotassiuni salt ( EDl'A) , and 0.5 international unit of Hrparin per ml, was used to wash out alveolar rnacro- phages. Cells were obtained essentially by the method described by Russczll & Rarer (16 ) with modification as described earlier ( 2 ) . The an- terior chest wall and pretraclieal musclcs were dissected away. A 14 gauge needle was forred into the trachea just below thr pharynx. One nil of the washing fluid was forced into thr lung and withdrawn after a few seconds without massage of the lungs. The washing was repeated twice. Cell suspensions from 5-~7 mice wrre pooled, cen- trifuged at 600 rpm for 10 minutes, and re- suspended in an appropriate volume of Eagle- Hanks Minimum Essential Medium ( M E h l ) . Sarnplrs macroscopically contaminated by red blood cells were discarded.

Cell rounts: Wet preparations were counted in a Burker chamber, quantitating the macrophages only. Prepartinns for differential counts were stained with Wright's stain. Viability of cells was examined by 'l'rypan blue staining, using 1 per cent solution of the stain.

Phngocytosis in sicspcnsion: The procedure used was in essentials similar to the method of Cohn & Morsr ( 1 ) , with modification as drscribed earlier ( 2 ) . Cells were dispersrd into 10 x 70 mm tubrs. Each tube contained ca. 4 ~8 x 10' macrophages in Eagle-Hanks MEhl at p H 7.2, with 20 pcr crnt normal mouse serum. Bacteria werr addrd in a ratio of about 5 -20 bacteria per niacropliagc. 'l'lie total volume in each tube was 1 1111. Control t u lm without cells were included in each experiment. The tubes were incubated in an incubator at 37" C with an end-to-rnd mixing at 40 rpm. At 30 and 60 minutes, samplcs were removed for the surviving bactrrial count. Extracrllular bactrria were counted by plating appropriate dilutions from the suprrnatant after differential centri- fugation a t 600 rpm for 5 minutcs. Totally viable bacteria were counted after brraking up the rna- crophages with 5 per cent saponin for 15 minutes at room trmperature, then plated in appropriate dilutions. All plating was done on cliocolatr agar.

Phagocytosis by monolayfJrs of mcrcrophngi,.s: The method employed was similar to that described by Whitby & Rorulry (18 ) with modification as de-

42

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scribed earlier ( 2 ) . Washed cell suspensions con- taining ca. 2-5 x 10:’ macrophages were pipetted into Leighton tubes in which a “flying coverslip” covered the entire bottom surface. Normal mouse serum was added to make a final 2 0 per cent concentration. The mixture were incubated at 37” C for 18 hours. After such treatment the macro- phages attached themselves to the surface of glass, while other crll types were dead or washed off. Bacterial suspension in Eagle-Hanks MEM a t pH 7.2 was added to make a final ratio, ca. 100 macrophages per bacterium. Normal mouse seruni was added in a final concentration of 20 per cent. The total volume was 1 ml. The tubes were in- cubated stationary for 1 hour, then the surviving bacteria were counted. Supernatant fluid was re- moved and plated in appropriate dilutions. The coverslips with the macrophage monolayers were also removed and washed with physiological sa- line three times, in order to remove bacteria not closely associated with the cells. A few drops of 5 per cent saponin were pipetted onto the co- verslips. After a few minutes the cowerslips were inverled and rubbed onto the surface of agar media to dislodge any macrophages and bacteria which were still attached to the surface. Colony counting was done after 18 hours’ incubation at 37” C.

The difference in numbers of bacteria recovered from test series and control series was attributed to the influence of phagocytic cells. The function of these cells was further divided into phagocytic action and intracellular killing, shown by the dif- ference in numbers of bacteria recovered from the extracellular fluid and from the destroyed phagocytic cells.

Sta!istical methods: Differences in mortality were analysed with a ,+test. To evaluate the phagocytosis data, Wilcoxon’s two sample test was used.

R E S U L T S

Since both bacterial strain and mouse strain were different from those employed in pre- vious studies ( 3 ) it was necessary to deter- mine whether the synergistic effect could be reproduced with the present system. Groups of mice were inoculated with lo6 egg in- fectious dose (EID) virus, 1-3 x 10‘ colony forming unit ( C F U ) bacteria, or with the combination of both agents, and mortality was recorded. In the present study, mortality after 10 days’ observation was higher in the H . influenza infected group (37 per cent) and in the group infected with both agents (83 per cent) than in the previous study (6

and 54 per cent). Mortality after virus in- fection only was 10 per cent. The synergistic effect was also clearly demonstrated with the present system.

PhagocytoJis by Macrophages from Infected Mice

Macrophages were obtained from mice 4 days after they had received virus inocula- tion. We have demonstrated earlier that the effect of virus infection on bacterial clear- ance and on gross lungpathology was max- imal with 4 days’ difference between inocula- tions. Wash-outs from 5-7 mice were pooled to make a total 4-8 x 10‘ macrophages per tube. An equal number of cells obtained from normal mice served as controls. In the first series phagocytosis was tested with cells in suspension. The mean of the results of 6 experiments are shown in Figs. 1 and 2. Fig. 1 presents the percentage of bacteria re- covered from the extracellular fluid. The proportion of bacteria eliminated from the experiments are shown in Figs. 1 and 2. Fig. 1 presents the percentage of bacteria re- from virus infected animals were less efficient than cells from normal animals. The differ- ence is significant a t 30 minutes ( p < 0.05) but not a t 60 minutes (0.05 < p < 0.1). Fig. 2 gives the percentage of bacteria recovered from the total mixture. Elimination of bac- teria from the total sample was attributed to the bactericidal action of macrophages. Again cells from virus infected mice were less efficient than cells from normal animals. The differences are significant (a t 30 min- utes: p < 0.01, a t 60 minutes: p < 0.05). The difference between extracellular and total count is an expression of the bacteria associated with the cells, probably phagocy- tosed, but not killed. This proportion of the viable bacteria recovered from virus infected cells was slightly larger than that from nor- mal cells, however, the difference was not significant ( p > 0.1).

In the next series, phagocytosis was con- ducted with macrophages attached to glass surfaces. Samples for colony counts were

43

Page 4: SYNERGISTIC EFFECT IN VIRAL-BACTERIAL INFECTION. : 2. Influence of Viral Infection on the Phagocytic Ability of Alveolar Macrophages

30 60 T I ME (min)

Fig. 1 . Comparison of the phagocytosis of Haenio- philus influenzae by suspensions of alveolar ma- crophages from normal mice and from mice in- fected with parainfluenza virus. The curves are the means of 6 experiments and show the num- bers of bacteria remaining in the extracellular fluid.

-normal cells .......... .virus infected cells

.....................

I I

30 60 T I M E (rnin)

F i g . 2. ’l‘hr bactericidal effcct of alveolar rnacro- phages from nornial and from parainfluenza viriis infected mice against HaCmophi1ii.i inf l i icnxt . . Tlir curves are thc. nicans of 6 esperirnrnts arid sliow the total numbers of bactrria recovrred from cells and extracellular fluid.

TABLE 1. Phagocytosis and Intracellular Killzng of Haemophilus influenzae by monolayers of Alurolar Macrophages f rom Normal nnd f rom Pnrainfliwnzn l ’ i r l i i Inf(7rtc.d M i c e

Phagocytic cells Per cent bacteria recovered

Extracellular fluid Macrophages lo ta l

Normal macrophages Virus infectrd macrophages

77.8 92.5

4.6 82.6 6.6 99.1

taken at the initiation and at the end of the experiments after 1 hour’s incubation. The mean of the results of 6 experiments are shown in Table 1. Normal macrophages were significantly more rfficient than virus in- fected ones in reducing the number of bac- teria in the extracellular fluid and in the total samples ( p < 0.05). The proportion of bacteria recovered from the macrophages was not significantly different.

These experiments suggest that the phago- cytic capacity of mouse alveolar macrophages is reduced after intranasal infection with parainflucnza virus, compared with rells

44

from normal micr. The ability to kill bac- teria already engulfcd dors not seem to b? influenced by the virus infcction.

Phagocytosis by Cclls 1iifrctr.d witlr Virus in uitro

A large pool of normal macropliages was divided into equal parts and cultured in Leighton tuhrs. After the attachment period, some tubes were infectvd with virus. othcr tubes were left uninfertrd for control. By this technique the cell populations in the infected tubes and in the rontrol tubes were

Page 5: SYNERGISTIC EFFECT IN VIRAL-BACTERIAL INFECTION. : 2. Influence of Viral Infection on the Phagocytic Ability of Alveolar Macrophages

completely homogeneous. In pilot expFri- ments phagocytosis was tested 4 days after virus inoculation. The infected cell popula- tion revealed a reduced phagocytic activity. The inhibitory effect was dependent on the concentration of the infecting virus (Fig. 3 ) . The minimum concentration which gave de- tectable effect was 10' EID, increasing to about 100 per cent inhibition when the in- fecting dose was 10" EID virus. In the fol- lowing phagocytosis experiments the con- centration of infecting virus was 10' EID per tube.

4 - 2,

- 2 0 ° , f e

Effect of T i m e Interval after Virus Inocu- lation on Phagocytosis

The development of in uitro infection is certainly different from that of in uiuo in- fection. The four-day interval employed in the pilot experiments is probably not com- parable to that after in uiuo infection be- cause the cells are or could be at different pliases of the infection. In a series of ex- periments we have investigated the effect of the length of time after in uiuo and in uitro

DOSE OF INFECTING VIRUS

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infection on the inhibition of phagocytosis. I t was also of considerable interest to de- termine the variation of virus effect on phagocytosis according to the time after in- fection and its correlation with gross patholo- gy of the lung and bacterial clearance rates when different time intervals between virus and bacterial infections were used.

The phagocytic capacity of macrophages at different times after in uitro infection was tested and related to the phagocytosis by normal cells originating from the same cell pool. The results of a typical experiment are shown in Fig. 4. The extent of inhibition at different times varied only slightly, but the tendency was consistent. The development was characterized by a maximal inhibition 24 to 48 hours after virus inoculation (max- imally 26 per cent) ; thereafter the capacity gradually returned to normal.

In the next series, macrophages were ob- tained from infected mice. Groups of mice were infected with lo6 EID virus on conse- cutive days. From 2 hours through 5 days after infection all groups were sacrificed, macrophages were obtained, and their phago-

2 hours 1 2 3 4 5 DAYS AFTER VIRUS INFECTION

Fig. 4 . Phagocytosis of Haemophilus in- fluenrae by alveolar macrophages at various times after in uitro infection with parainfluenza virus.

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-

:: D A Y S AFTER VIRUS INFECTION

Fig . 5. Phagocytosis of Hocrnophilus in- fluc,nzar by alveolar macrophages at various times after in uiuo infection with parainfliienza virus.

cytic capacity was tested and compared with that of normal control cells. The results of a typical experiment are shown in Fig 5. The extent of inhibition at different times after in viuo infection was closely correlated with that after in uitro infection at corresponding times. Maximal reduction was recorded 24 hours after virus inoculation. Unfortunately the 2 days’ samples were not reprcsentativc in eithcr series completed.

Tht, Effect of Time Interval on Mortality

To evaluate further the effect of the length of time between viral and bacterial inoculation, mortality rates were determined. Groups of mice were infected with virus. and a t various times from 2 hours through 5 days later with bacteria. Mice infected with one agent only served as controls. The groups were observed for 10 days and mortality was recorded. The results arc given in Fig. 6. Each group consisted of 36 to 40 mice. Com- pared with the control groups, mortality was not significantly enhanced when the combi- nation of infections were given 2 hours apart. I t was, however, significantly higher ( p < 0.01) i f the bacterial infection followed the virus 1 through 5 days later. Among these groups again mortality was significantly higher ( p < 0.01) in the group infected at a 4 days’ interval, when compared with thc rest. The difference could also be expressed in terms of the length of survival. Main sur- vival in days a t 2 hours’ interval: 7.51 days; a t one day: 5.04; at two days: 4.97; at three

46

days: 5.02; at four days: 2.02 and a t fivc days: 4.05 days. I n the present system of dual infection the data concerning both mor- tality and length of survival strongly indicate that the synergistic effect is most pronounced when the agents are inoculated 4 days apart.

Effcct of Sc,runi Factors on Phagocptosi~

In all tlic experiments described, 20 per cent normal mouse serum was included in the mixture. Exclusion of scrum reduced thc phagocytic activity to practically nil. Scrum from mice previously immunized with and containing agglutinating antibody against H . influcnzar~ incrcased the phagocytic activity. No diffcrcnce was obsrrved when thc op- sonizing cffect of normal scrum was rom- parcd with that of serum from micc im- munized with parainflucnza virus.

D I S C U S S I 0 N

The present data indicate that infcction witli parainfluenza 1 virus inhibits the phagocytic activity of mice alveolar macropliages.

Inhibitory effect on the antibactcrial ac- tivity of pliagocytic cells by virus infection has been dcscribed earlier. Influenza and mumps virus infection reduced tho pliago- cytic action of guinea pig leucocytes ( 13). Ginsberg et al. (4. 5, 6 ) also reported in- hibition of phagocytosis by guinca pig Icuco- cytes after infection with influenza virus. The mechanism of inhibition was postulated

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Per cent

90

I 80 -

70 -

60 -

50 -

40 -

30 -

20 -

10

4 days

I

Fours bacteria only x d l 1- virus onty e---

/ I 1 1 1 1 1

1 2 3 4 5 6 7 8 9 10 Days after bacterial infection

F i g . 6. The influence of the interval between inoculation with parainfluenza virus and Huemophilux influenzae on the mortality of mice. The curves show the cumula- tive death rates.

to be duc to a block in the energy-yielding mechanisms of virus infected cells. Inhibition of phagocytic activity of horse and guinea pig leucocytes by coxackie viruses was ob- served by Kantoch & Dubowsku-lnglot ( 10). The effect was due to the action of un- inactivated virus associated with the cells, and it was dependent on the concentration of the virus and on the length of its action.

Some authors also demonstrated an in-

hibitory effect on phagocytosis during com- bined infection of the respiratory tract. Wil- son et ul. (19) investigated the role of hu- moral and cellular immunity in dual infec- tion of monkeys by influenza virus and Streptococcus huemolyticus. A depression of phagocytic properties of the circulating gra- nulocytes was demonstrated, usually within 24 to 48 hours after virus infection. A delay in migration of polymorphonuclear leuco-

47

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cytes and slow phagocytic action of pul- monary macrophages during combined in- fection of mice with influenza virus and pneumococci were observed by Harford & H a r a (9) . Similarly, a reduction of pliago- cytosis by guinea pig macrophages and granulocytes after influenza virus infection was reported by Saroyrr & W o o d (17 1. How- ever, the significance of their finding for the pathogenesis of the dual infection was doubted. K l r i n et al. (12 ) demonstrated that the bactericidal ability of mouse lung was reduced after reovirus infection. This change was attributed to an effect on the alveolar macrophage system.

The inhibitory effect in the present series could be demonstrated with macrophages when in suspension or when they were at- tached to solid surfaces. We attempted to avoid alternation of the cells employed in the phagocytosis experiments in suspension, during preparation. They were treated care- fully with a minimum of handling, and each experiment was completed within 2 hours after removal of the cells from the organism. A significant proportion of polymorphonu- clear granulocytes was regularly seen in the cell population from virus-infected animals. These cells were included in the phagocytosis experiments in addition to the macrophages, but only the latter were quantitated. We have no data on the extent of participation of leucocytes in the phagocytosis. However, many of them were non-viable by the trypan blue staining method, and could not have phagocytic activity. If the granulocytes were of considerable significance one would expect an increased difference between normal and virus infected cell populations when the ac- tivity of granulocytes are eliminated, e . g. when macrophages are tested in pure culture. The reduction of the phagocytic capacity, however, was more marked when the cells were in suspension which contained poly- morphonuclear leucocytes in addition to macrophages than when cells on glass sur- faces were pure cultures of macrophages. Pavlousky ( 1 5 ) reported that the partic- ipation of leucocytes in the phagocytosis of

48

cytoplasrnatic inclusions during influenza in- fection was negligible in comparison with the much higher one of macropliagrs.

The surface technique allowed t l i r pcr- formance of phagocytosis experiments with practically pure cultures of macrophages. Other cell typcs werr dead or did not at- tach themselves to the surfaces. The proccass, Iiowever, involved culturing in ritro for sev- era1 hours, a process which niiglit altcxr somt’ of the preperties of the cells. Extcnded cul- turing reduced slightly tlic phagocytic ac- tivity. Controls of identical “in uitro age” were included in all experiments. I t is doubt- ful whether the surface phagocytosis phe- nomenon, described by W o o d (20) . has any significance on the results of thc prcwrit ex- periments.

The surface teclrniqur also made i t pos- sible to investigate the effect of in uitro in- fection of cells. Although removal of crlls from their natural environment rniglit alter their properties it makes it possible to se- parate the direct action of thca virus on the cells from possiblca indirect mechanisms. In uitro infection also inhibited the pliagocytic activity of macrophages to an extrnt similar to that in in r k ) infection. Serum opsonir activity was not affected by previous ex- perience with virus. The data suggest tliat the mechanism by which the virus influences the phagocytosis is one acting directly on the phagocytic cells. Tlic- cellular site of virus action was also pointed out by Fixher & Ginsbrrg (4, 5) and by Kautock & Duhow- sku-lnglot ( 10) . The mcdranism of inhibi- tion on the subcellular level in the prescnt system remains to bc invcstigated.

The length of time between primary and secondary inoculations seems to liave critical influence on the development of dual in- fection. Mortality did not increase whrm thr agents were given 2 hours apart, as compared with the death rate aftcar infection with either agent alone. The mere presence of both agents in the organism does not en- Iiancr mortality. This indicates that the sy- nergistic effect is a result of a time dependent process. Several data indicate that the in-

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teraction is most efficient when the time in- terval between primary and secondary inocu- lation is 4 days. I t is reasonable to assume that total host defence is maximally reduced about 4 days after virus infection. ‘The total host defence is a result of the combination of a number of defence factors. The capacity of the defence factor of factors critical in the development of the disease in dual in- fection is probably maximally reduced a t this time. The present data indicate that in- hibition of phagocytosis is at its largest ex- tent at an earlier phase, 24 to 48 hours after virus infection. I t seems to be questionable therefore whether the reduction of phago- cytic activity is the critical factor, at least that it is the only one affected. A similar conclusion was presented by Sawyer & Wood (17), using influenza virus as a model. The lack of correlation in time makes it necessary to postulate that other local defense factors must be in operation and probably they are critical for the development of combined in- fection. It cannot be ruled out that phago- cytosis has an important role. From the de- sign of present experiments, however, it is not possible to determine how much the in- hibition of phagocytic capacity contributes to the total reduction of the capacity of host defence.

This study has been supported by the Norweginn Pasteur Foundation.

Present address: Kapt. Wilhelnisen og Frues Bak- teriologiske Institutt, Rikshospitalet, Oslo, Norway.

R E F E R E N C E S

Cohn, Z . A . & Morse, S. E.: Interaction be- tween rabbit polymorphonuclear leukocytes and staphylococci. J. Exp. Med. 110: 419- 443, 1959. Degrk, M.: Phagocytic and bactericidal ac- tivity of peritoneal and alveolar macrophages from mice. J. hled. Microbiol. 2: 353-357, 1969. l l egrk , M . & Glasgoru, L. A , : Synergistic ef- fect in viralbacterial infection: 1. Combined infection of the respiratory tract of mice with parainfluenza virus and Hemophilus influenza. J. Infect. Dis. 118: 449462, 1968.

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